Our long-term goal is to investigate the in vivo requirements for inducing GVHD. T-cell activation and expansion occur as a result of T-cell receptor ligation and costimulatory signals. It now is apparent that there are families of costimulatory molecules some of which deliver positive and others negative signals to T-cells. We hypothesize that the purpose of these pathways is to regulate different phases of T-cell response and provide some redundancy to protect the host if one system fails. We hypothesize that CD28/CTLA-4:B7 and CD40 ligand (CD40L)/CD40 interactions regulate the initiation phase of the T-cell response while the recently described inducible costimulatory molecule (ICOS) regulates the memory/effector phase of GVHD. We will determine whether the ICOS/ICOS-L pathway is additive to the effects of known positive (CD28/B&; CD40L/CD40) regulators critical in GVHD. We also hypothesize that the engagement of PD-1 (programmed cell death-1) on activated T-cells by its ligand, PD-L1 and PD-L2, will inhibit GVHD generation. We further hypothesize that the negative signals delivered via PD-1 are not redundant with the CTLA4/B7 pathway. T-cells also can directly inhibit the function of other T-c3lls via mechanism(s) not fully understood. The most well-studied are CD4+25+ regulatory cells. We have shown that these cells are required for in vitro tolerance to alloantigen by costimulatory pathway blockade and are important negative regulators of GVHD lethality in vivo. Since CD4+25+ or CD4+25- cells are express ICOS, PD-1 as well as CD28, CTLA-4 and CD40L, we propose to determine whether CD4+25+ or CD4+25- cells are differentially affected by positive and negative costimulatory pathway blockade. We are in a unique position to perform these studies by having extensively investigated the role of T-cells costimulation and CD4+25+ cells in GVHD, access to unique reagents for targeting of these pathways, and collaborators who are experts in these areas. Collectively, our studies will improve the understanding of the pathophysiology of GVHD induction as well as the development of approaches to block GVHD generation which can be translated into humans.